Humans who ingest Listeria monocytogenes (Lm)-contaminated food develop infections that range in severity from mild, self-limiting gastroenteritis to life-threatening sepsis and meningoencephalitis. Strain lineage, dose, and host susceptibility factors are all likely to influence infection outcomes, but our understanding of the intestinal phase of infection is still severely limited. To overcome this obstacle, we recently developed a murine model of foodborne listeriosis that can be used to study both virulence determinants and immune responses in animals that vary in their susceptibility or resistance to infection. Using this model, we found that the vast majority of Lm in the gut were extracellular. This result was unexpected, because Lm are facultative intracellular pathogens and most research efforts over the past few decades have focused on Lm factors that promote invasion, survival and growth in cultured mammalian cells. Mutant Lm that were unable to replicate inside host cells established infection in the murine gut normally, and persisted for a few days, but had a severe defect in disseminating from the intestinal lamina propria (LP) to the mesenteric lymph nodes (MLN). These observations suggest that intracellular growth is not needed to cause gastroenteritis; however, further systemic spread requires replication inside an as-yet-unidentified cell type in the gut. The central hypothesis of this proposal is that exponential replication in a permissive cell type is needed to evade innate clearance mechanisms in the gut, and to increase the number of Lm above a critical threshold that can promote dissemination by one of three distinct mechanisms. In Aim 1, ex vivo flow cytometry and microscopy approaches will be used to identify all of the cell types that can be productively infected with Lm in the ileum, colon, and the MLN that drain these tissues. In Aim 2, we test the hypothesis that Lm can use one of three potentially redundant mechanisms to spread from the gut LP to the MLN: 1) attached to migratory cells that are not efficiently invaded; 2) ?stealth transport? in the cytosol of other migratory cells; and 3) extracellular Lm trafficking free-flowing in lymphatic vessels. Building on the knowledge gained from Aims 1 & 2, Aim 3 seeks to address the paradox that mechanisms exist for Lm to spread extracellularly, yet intracellular replication is needed at some point in the infection cycle for efficient dissemination to the MLN. The replication rate in the gut will be altered by eliminating the cell types permissive for intracellular growth, and the clearance rate will be altered by depleting neutrophils and inflammatory monocytes. A key strength of this proposal is the interdisciplinary nature of the approaches that capitalize on recent advances in understanding mononuclear phagocyte differentiation pathways in the steady state and applying those findings to a physiologically relevant infection model. This study will advance the field by revealing how Lm survive and replicate in the gut and by identifying the bottlenecks they face in order to cause systemic disease.